Tooth development is a complex morphogenetic process that involves both intrinsic genetic cues and epigenetic environmental signals. Recent studies have begun to reveal some of the regulators of tooth morphogenesis, including homeodomain transcription factors and bone morphogenic proteins (BMPs). The focus of this application is a novel homeodomain gene that is linked to Rieger syndrome. Rieger syndrome is characterize by morphogenetic abnormalities that include dental hypoplasia. The Rieger gene, now termed Pitx2, is a member of the bicoid-like family and has transactivation activity. It is the earliest known marker in the tooth primordia and is believed to play a key role in early tooth morphogenesis. However, the mechanisms that regulated Pitx 2 expression and function are not known. For example, our preliminary data on Pitx2 transactivation suggests a novel inhibitory role for the C-terminal domain. Furthermore, Pitx2 expression requires an unknown mesenchymal signal and additional factors are clearly needed for full transactivation activity. A major objective of this application will be to answer the question, what are the factors and signals that regulate Pitx2 function? We propose to first establish a working model of Pitx2 functional domain. In this context we will ask how does the C- terminal region inhibit transactivation and how do the naturally occurring Rieger mutations affect Pitx2 function? We will then examine the mechanisms controlling Pitx2 expression and function. In the first set of experiments we will examine control of Pitx2 by signaling molecules. Since tooth formation relies on epithelial-mesenchymal interactions, we propose to test the hypothesis that the extracellular signals BMP-4 and Sonic hedgehog (Shh) regulate Pitx2 expression in explant cultures. We will also test whether those agents activate Pitx2 promoter activity ion a model cell line. In the second set of experiments, we will ask if Pitx2 activity is controlled by physical interactions with oral epithelial transcription factors, as suggested by our preliminary studies using the Pit-1 protein. We will test interactions with known candidates that are co-expressed with Pitx2 in the dental epithelia, such as the Msx2 homeodomain protein. This approach will also enable us to identify novel interacting partners. We will then test the hypothesis that these protein interactions are important for Pitx2 function. These studies will provide the foundation for a better understanding of the molecular control of early tooth development.